ABSTRACT Excision of introns from precursor messenger RNA by the spliceosome is a critical step in almost all human gene expression. This process is highly regulated, integrally linked with the transcription of genes and other processing events, such as polyadenylation and nucleotide modification. The mechanism by which the spliceosome recognizes the exact sites for the chemical events and how the reactions are catalyzed are not well understood. The long-term goals of this project are to understand interactions and rearrangements between spliceosome components and the RNA ligands that are substrates for the catalytic reactions. Ample evidence argues for multiple rearrangements of factors and multiple recognition events at the branch site. Investigation of these events ? which are not understood mechanistically ? will elucidate interactions and rearrangements among core components and may serve as a paradigm for rearrangements in the spliceosome and in other RNP machines. This proposal focuses on mechanisms by which spliceosomal dynamics impact splicing fidelity. Experiments will first investigate binding and positioning of the 3'SS-UAG onto the spliceosome. Binding of the spliceosome to the 3'SS is critical for intron definition, for spliceosome assembly, and for splicing catalysis. Yet, nothing is known of spliceosome?3'SS-UAG interaction, other than the early interaction with U2AF. Here we use an `orthogonal spliceosome' (second-copy, reverse-engineered, designer spliceosome) that we have developed in yeast, to identify both the 3'SS binding site for second-step catalysis and a `loading site' for 3'SS on the assembling spliceosome. Second, two large gaps in our understanding of RNA biology are the identification of RNAs between 50 and 200 nts, which are missing in almost all modern-day sequencing datasets, and the bioinformatic analysis of repetitive sequences ? the snRNAs represent both. We have identified novel U2 snRNA variants that are expressed differentially in cells, and we will investigate the components, function, and substrates of novel U2-variant spliceosomes.